The remote substrate binding subsite -6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism.

H.Leemhuis,J.C.Uitdehaag,H.J.Rozeboom,B.W.Dijkstra,L.Dijkhuizen.

ABSTRACT

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-,
beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6,
7, or 8 glucose residues, respectively) from starch. Nine substrate binding
subsites were observed in an x-ray structure of the CGTase from Bacillus
circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is
conserved in CGTases, suggesting its importance for the reactions catalyzed by
the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F,
G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had
decreased k(cat) values for beta-cyclodextrin formation, as well as for the
disproportionation and coupling reactions, but not for hydrolysis. Especially
G179L, G180L, and G179L/G180L affected the transglycosylation activities, most
prominently for the coupling reactions. The results demonstrate that (i) subsite
-6 is important for all three CGTase-catalyzed transglycosylation reactions,
(ii) Gly-180 is conserved because of its importance for the circularization of
the linear substrates, (iii) it is possible to independently change cyclization
and coupling activities, and (iv) substrate interactions at subsite -6 activate
the enzyme in catalysis via an induced-fit mechanism. This article provides for
the first time definite biochemical evidence for such an induced-fit mechanism
in the alpha-amylase family.

Selected figure(s)

Figure 1.
Fig. 1. A, schematic overview of the interactions between
the B. circulans strain 251 CGTase and a maltononaose substrate.
For clarity not all interactions at subsites +1, 1, and 2 are shown
(21). B, maltononaose (black) conformation in the active site of
CGTase. The arrow indicates the cleavage site. B was made using
Swiss-PDB-Viewer (41).

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